European Journal of Pharmacology

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Cardiovascular pharmacology

Vasopressin attenuates ischemia – reperfusion injury via reduction of oxidative stress and inhibition of mitochondrial permeability transition pore opening in rat hearts

Afshin Nazari

^a

, Seyed Shahabeddin Sadr

^b,c

, Mahdieh Faghihi

^b

, Yaser Azizi

^b

, Mir-Jamal Hosseini

^d

, Naser Mobarra

^e

, Asadollah Tavakoli

^f

, AliReza Imani

^b,n

aDepartment of Physiology, School of Medicine, Lorestan University of Medical Sciences, Khorramabad, Iran

bDepartment of Physiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

cElectrophysiology Research Center, Neuroscience institute, Tehran University of Medical Sciences, Tehran, Iran

dZanjan applied Pharmacology Research Center, Zanjan University of Medical Sciences, Zanjan, Iran

eDepartment of Biochemistry and Biophysics, Metabolic Disorders Research Center, School of Medicine, Golestan University of Medical Sciences, Gorgan, Iran

fDepartment of Physiology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

a r t i c l e i n f o

Article history:

Received 10 March 2015 Received in revised form 4 April 2015

Accepted 8 April 2015 Available online 17 April 2015 Keywords:

Heart

Ischemia/reperfusion Vasopressin

Mitochondrial swelling Free radical

a b s t r a c t

Aim of this study was to investigate the involvement of the mitochondrial permeability transition pore (MPTP) and oxidative stress in the cardioprotective effect of vasopressin (AVP) on ischemia/reperfusion (I/R) injury. Anesthetized male wistar rats were subjected to regional 30 min ischemia and 120 min reperfusion and randomly divided into nine groups: (1) Control; saline was administered intravenously before ischemia, (2) vasopressin was administrated 10 min prior to ischemia, (3, 4) Atractyloside as MPTP opener, was injected 5 min prior to reperfusion without and with vasopressin, (5, 6) Cyclosporine A as a MPTP closer, was injected 5 min prior to reperfusion without and with vasopressin, (7) mitochondria were isolated from control group and CaCl2was added as MPTP opener and swelling inducer, (8) isolated mitochondria from Control hearts was incubated with Cyclosporine A before adding the CaCl2(9) CaCl2

was added to isolated mitochondria from vasopressin group. Infusion of vasopressin decreased infarct size (18.671.7% vs. control group 37.672.4%), biochemical parameters [LDH (Lactate Dehydrogenase), CK-MB (Creatine Kinase-MB) and MDA (Malondialdehyde) plasma levels, PAB (Prooxidant–antioxidant balance)] compared to control group. Atactyloside suppressed the cardioprotective effect of vasopressin (32.571.9% vs. 18.671.7%) but administration of the Cyclosporine A without and with vasopressin signiﬁcantly reduced infarct size to 17.774% (Po0.001) and 22.773% (Po0.01) respectively, vs.

37.672.4% in control group. Also, vasopressin, similar to Cyclosporine A, led to decrease in CaCl2- induced swelling. It seems that vasopressin through antioxidant effect and MPTP inhibition has created a cardioprotection against ischemia/reperfusion injuries.

1. Introduction

Cardiac ischemia occurs frequently in critically ill patients and is associated with increased mortality (Lim et al., 2010). Cardiac preconditioning represents the most potent and consistently reproducible method of rescuing heart tissue from undergoing irreversible ischemic damage. Unfortunately, the clinical value of ischemic preconditioning (IPC) is limited. None of the several identiﬁed pharmacologic agents that appear to limit reperfusion injury is available for clinical use (Yang et al., 2004). Vasopressin or arginine vasopressin (AVP) is essential for cardiovascular homeostasis (Holmes et al., 2001). Circulating levels of

AVP, which are elevated during hypovolemia and during cardiac stress, mediate important physiological functions such as osmotic regulation, vasoconstriction, and release of adrenocorticotropic hormone (ACTH) (Zhu et al., 2013). In the previous study we have shown that AVP (0.03mg/rat) has protective effects on ischemia/reperfusion (I/R) induced myocardial injury in rat heart (Nazari et al., 2011). Interest- ingly, other study conﬁrmed the protective effects of AVP in low dose on myocardial injury of the ischemic reperfused heart (Pelletier et al., 2013; Zhu et al., 2013). Also vasopressin infusion is cardioprotective in models of myocardial ischemia (Okamura et al., 1999) and in patients with postcardiotomy shock (Dunser et al., 2002). Moreover, we reported that AVP (0.03mg/rat) provides cardioprotection against heart I/R injury by its anti-oxidant action (Nazari et al., 2011). However, the exact mechanisms of cardioprotection of AVP remain poorly understood.

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European Journal of Pharmacology

nCorresponding author. Tel./fax:þ98 21 66419484.

E-mail address:[email protected](A. Imani).

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Mitochondria are the most important cellular sources of reactive oxygen species production and are particularly suscepti- ble to oxidative stress (Sastre et al., 2003). Indeed, conditions associated with post-ischemic reperfusion, such as reactive oxygen species accumulation, pH normalization and increases in [Ca²^þ], create an ideal scenario for mitochondrial permeability transition pore (MPTP) opening (Zoratti and Szabo, 1995). Opening of the MPTP may result in mitochondrial swelling, collapse of mitochondrial membrane potential, uncoupling of mitochondrial oxidative phosphorylation and cytochrome C release, leading to both necrosis and apoptosis. It was demonstrated that preventing MPTP opening may be protective in isolated cardiomyocytes (Nazareth et al., 1991) and perfused hearts (Grifﬁths and Halestrap, 1993). In addition, modulation of MPTP opening has been observed in cardioprotection by both preconditioning (Xu et al., 2001) and post-conditioning (Argaud et al., 2005) of the heart. Inhibition of MPTP opening also exerts cardioprotection against I/R injury in mice induced in vivo (Wang et al., 2005).

The purpose of the present study was to determine whether AVP protects the heart at reperfusion through a mitochondrial pathway, speciﬁcally via inhibition of MPTP opening.

2. Materials and methods

Male Wistar rats (Weighing 280–310 gr) housed under stan- dardized conditions 12-h light/dark cycle, 20–221C ambient temperature and 40–50% humidity with free access to fed standard rat chow and tap water. All animal care and experiments were conducted in accordance with the institutional guidelines of Tehran University of Medical Sciences (Tehran, Iran).

2.1. Surgical preparation

Anesthesia was achieved by administration of sodium thiopental (60 mg/kg, i.p.). Body temperature was maintained at 37711C. After a tracheotomy, all rats were ventilated with air and oxygen mixture by Parvalux rodent respirator (15 ml/kg stroke volume and 60–70 Breaths/

min). The right carotid artery was dissected and a heparinized saline (100 U/ml) ﬁlled polyethylene-tubing catheter (PE-50) was inserted into the artery for blood sampling and hemodynamic monitoring. The femoral vein was cannulated to inject Evans blue and drugs. Lead-II electrocardiogram (ECG) and arterial hemodynamic parameters were continuously monitored and recorded throughout the experiment, using a computerized data acquisition system (ML750 PowerLab/4sp, AD Instruments). 10 min prior to the end of reperfusion period, the carotidal catheter was advanced to the Left Ventricle (LV) to record the functional parameters of LV (Smith et al., 1979).

Rats were given heparin (200 IU/kg, i.v.), and then the chest was opened by a left thoracotomy in the fourth rib to expose the heart. The pericardium was incised and a 6–0 silk suture was placed around the left anterior descending coronary artery (LAD) close to its origin. Both ends of the suture were passed through coronary ligator. Heart rate and blood pressure were allowed to stabilize for 15 min before the intervention protocols. Applying tension to the suture by ligator caused regional ischemia, and reperfusion was achieved by releasing the tension on the ligature.

Ischemia was conﬁrmed by ST elevation in ECG, or cardiac cyanosis subsequent decrease in blood pressure, and reperfusion was conﬁrmed by epicardial hyperemia.

2.2. Experimental protocol

After a stabilization period following the surgical preparation, basal hemodynamic parameters were measured for 15 min before drug administration and the heart of all animals was subjected to

30 min ischemia and 120 min reperfusion (Fig. 1). Rats were randomly divided into nine groups: (1) Control; saline was administered intravenously before ischemia, (2) AVP0.03; vasopressin 0.03mg/rat was infused within 10 min prior to ischemia (n¼13), (3, 4) Atr and AVPþAtr; Atractyloside (5 mg/kg, i.v.), as a MPTP opener, was injected 5 min prior to reperfusion without and with the effective dose of AVP 0.03mg/rat into two different groups (n¼13), (5, 6) CsA and AVPþCsA Cyclosporine A (CsA, 5 mg/kg, i.v.), as a MPTP closer, was injected 5 min prior to reperfusion without and with the effective dose of AVP (0.03mg/rat) into two different groups (n¼13). (7) Control–CaCl2; mitochondria were isolated from control group and 200mmol/l CaCl2 was added to induce MPTP opening and swelling (n¼4), (8) CsA–CaCl2; an aliquot of mitochondria from Control hearts was incubated with 1mmol/l CsA for 2 min before the addition of 200mmol/l CaCl2

(n¼4) (9) AVP–CaCl2; mitochondria were isolated from AVP0.03 group and 200mmol/l CaCl2was added to examine mitochondrial swelling (n¼4).

2.3. Hemodynamic functions

Arterial blood pressure and heart rate (HR) were continuously monitored and recorded throughout the experiment. Left ventricular hemodynamic parameters such as Left ventricular End- Diastolic Pressure (LVEDP), left ventricular developed pressure [LVDP¼LVSP (Left ventricular systolic pressure)LVEDP], maximum rise and fall of LV pressures (þdp/dt and dp/dtrespec- tively) and RPP (Rate pressure product¼LVDPHR) were recorded at 10 min of end reperfusion.

2.4. Cardiac area at risk and infarct size determination

At the end of reperfusion, the coronary artery was reoccluded and 2 ml of Evans blue (2%) was injected intravenously to the femoral vein. Then, the heart was excised, cut into 2 mm slices. All slices were incubated with a 1% 2,3,5-triphenyltetrazolium chloride (TTC, in 0.1 M phosphate buffer, pH 7.4) stain for 15 min at 371C, to visualize the infarct area. Then they were ﬁxed in 10%

formalin to enhance the contrast of the Evans blue and TTC staining. Both surfaces of each section were scanned using Photo- shop program (Adobe Systems, version 7.0). Total area at risk was expressed as a percentage of the left ventricles (AAR/LV). Infarct size was expressed as a percentage of the area at risk (IS/AAR).

2.5. Biochemical analysis

Blood samples were collected at the end of reperfusion for measure- ment of the cardiac enzymes, including creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH) and Malondialdehyde (MDA). The heparinized samples were centrifuged at 5000g, for 15 min, and the plasma was removed and stored at 701C until the time they were assayed.

The activity of CK-MB and LDH were analyzed using commercial kits (Pars Azmoon, Iran) by employing an autoanalyzer (Roche Hitachi Modular DP Systems, Mannheim, Germany). MDA content of samples was determined spectrophotometrically using a modiﬁcation of the assay described bySchuh et al. (1978).

2.6. Prooxidant–antioxidant balance (PAB) assay

A modiﬁed PAB was applied based on a previously described method (Alamdari et al., 2007). The standard solutions were prepared by mixing varying proportions (0–100%) of 250mM hydrogen peroxide with 3 mM uric acid (in 10 mM NaOH). 60 mg TMB (3,3⁰,5,5⁰-Tetramethylbenzidine) powder was dissolved in 10 ml DMSO; for preparation of TMB cation, 400ml of TMB/DMSO was added in 20 ml of acetate buffer [0.05 M buffer, pH 4.5], and then 70ml of fresh chloramine T (100 mM) solution

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was added into this 20 ml, mixed well, incubated for 2 h at room temperature in a dark place; 25 U of peroxidase enzyme solution was added into 20 ml TMB cation, dispensed in 1 ml and put at201C; in order to prepare the TMB solution 200ml of TMB/DMSO was added into 10 ml of acetate buffer [0.05 M buffer, pH 5.8]; the working solution was prepared by mixing 1 ml TMB cation with 10 ml of TMB solution, incubated for 2 min at room temperature in a dark place and immediately used. Ten microliters of each sample, standard or blank (distilled water) were mixed with 200ml of working solution, in each well of a 96 well plate, which was then incubated in a dark place at 371C for 12 min;

at the end of the incubation time, 100ml of 2 N HCl was added to each well; and measured in an ELISA reader at 450 nm with a reference

wavelength of 620 or 570 nm. A standard curve was provided from the values relative to the standard samples. The values of the PAB are expressed in arbitrary HK units, which are the percentage of hydrogen peroxide in the standard solution. The values of the unknown samples were then calculated based on the values obtained from the above standard curve.

2.7. Preparation of mitochondria

Mitochondria were isolated from male rats by differential centrifugation with some modiﬁcations (Ghazi-Khansari and Mohammadi-Bardbori, 2007). In brief, after deep anesthesia, the

Fig. 1.Illustration of the experimental groups. Animals in control group were subjected to 30 min ischemia followed by 120 min reperfusion and saline was administered intravenously before ischemia, (group 2) AVP0.03; effective doses of vasopressin (0.03μg/rat) was infused within 10 min prior to ischemia, (groups 3, 4) Atr and AVPþAtr;

Atractyloside (5 mg/kg, i.v.), as a MPTP opener, was injected 5 min prior to reperfusion without and with the effective dose of AVP (0.03mg/rat) into two different groups, (groups 5, 6) CsA and AVPþCsA; Cyclosporine A (CsA, 5 mg/kg, i.v.), as a MPTP closer, was injected 5 min prior to reperfusion without and with the effective dose of AVP (0.03mg/rat) into two different groups. NS, normal saline; TTC, triphenyltetrazolium chloride (n¼13).

Table 1

Hemodynamic parameters.

Groups Baseline End of ischemia 30⁰ End of reperfusion 60⁰ End of reperfusion 110⁰

HR SAP HR SAP HR SAP HR SAP

Control 308712 10975 290715 9775 269714^a 9274^a 233712^b 7675^b

AVP0.03 312718 11077 304714 9775 293715 8772^a 293719^c 8575^b

Atr 376716 12776 354713 11973 351722^c 10075^a 357713^d 9577^b

AVPþAtr 334711 12875 338710 11774 340711^c 10477^e 331712^d 84711^b

CsA 334727 12975 316725 11076 322717 11275^c 305714^f 9575^a

AVPþCsA 340710 12473 324718 10874^a 324715 10775^a 314715^f 9675^b

The values are mean7S.E.M. HR, heart rate (beats/min); SAP, systolic arterial pressure (mmHg); AVP, arginine vasopressin; Atr, Atractyloside; CsA, Cyclosporine A.

aPo0.01 vs. Its baseline within group.

bPo0.001 vs. Its baseline within group.

cPo0.05 vs. Control.

dPo0.001 vs. Control.

ePo0.05 vs. Its baseline within group.

fPo0.01 vs. Control.

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hearts immediately excised and AAR (area at risk) zone from LV was minced by small scissor in an ice cold manitol solution containing 0.255 M D-manitol, 74 mM sucrose and 0.2 mM ethy- lenediaminetetraacetic acid (EDTA) (pH¼7.4). Then the minced tissue was gently homogenized in a glass homogenizer with a Teﬂon pestle and then centrifuged at 1500gfor 10 min at 41C to remove nuclei, unbroken cells and other non-subcellular tissues.

The supernatants were centrifuged at 10,000gfor 10 min at 41C.

The pellet was resuspended in 1 ml of respiration buffer (70 mM sucrose, 230 mM manitol, 3 mM HEPES, 2 mM Tris–phosphate, 5 mM succinate and 1mM of rotenone) (pH¼7.4) for the determination of mitochondrial swelling.

2.8. Protein concentration

Mitochondrial protein concentrations were determined using the method developed byBradford (1976).

2.9. Mitochondrial swelling assays

Opening of the MPTP causes mitochondrial swelling and the change in mitochondrial volume due to colloidal osmotic effects of soluteﬂux into and out of the mitochondrial matrix was measured by monitoring the absorbance at 520 nm (A520) as described (Zhao et al., 2010). Brieﬂy, isolated mitochondria (approximately 500mg) in 1 ml of respiration buffer were pre-incubated at 301C for 10 min. After a 10 min equilibration period, 200 mmol/l CaCl2

was added to induce MPTP opening (Baines et al., 2003). Absor- bance was measured spectrophotometrically at 520 nm for 21 min at 3 min time intervals.

2.10. Materials

All chemicals used were obtained from Sigma Chemical Co.

2.11. Statistical analysis

Statistical analysis of arterial hemodynamic parameters within groups was performed with repeated measures ANOVA followed by Tukey's test and One-way ANOVA followed by Tukey's test was used to determine statistical significance in all other cases. All data were expressed as mean7S.E.M. Statistical significance was defined as Po0.05.

3. Results

3.1. Hemodynamic functions

Tables 1 and 2demonstrate the time course of heart rate and systolic arterial pressure (SAP) during the experiments. There were

no significant differences among groups at baseline before treat- ment. At the end of 60 min and 120 min reperfusion, heart function was significantly decreased in control group, as indicated by SAP and HR as compared with its baseline. Atractyloside and Cyclosporine A in (AVPþAtr) and (AVPþCsA) groups significantly prevented the decrease in HR at the end of 60 min and 120 min reperfusion compared to their baseline, but had no effect on SAP.

SAP not only decreased in CsA group at the end 60 min reperfusion but also significantly increased that compared to control group. As shown inTable 3, Atr and AVPþCsA caused significantly increasing in RPP compared to control. Moreover Atr caused significantly increasing in dp/dt compared to control. There were no significant differences of intraventricular parameters between any other groups.

3.2. Area at risk and infarct size measurements

There were no signiﬁcant differences in AAR/LV among groups.

Infarct size was 37.672.4% in control group, whereas AVP 0.03mg/

rat signiﬁcantly reduced infarct size to 18.671.7 vs. control group (Fig. 2). The reduction in infarct size by AVP 0.03 was abolished by Atractyloside infusion in AVPþAtr group as compared to AVP0.03 (32.571.9% vs. 18.671.7%) and Cyclosporine A administration in CsA and AVPþCsA groups signiﬁcantly reduced infarct size to 17.774% (Po0.001) and 22.773% (Po0.01) respectively, vs.

37.672.4% in control group and returned infarct size as seen as in AVP0.03.

3.3. Biochemical analysis 3.3.1. LDH and CK-MB activity

Compared to control group, administration of AVP 0.03mg/rat could prevent elevation of LDH activity in plasma after ischemia/

reperfusion injury. Also AVP 0.03 signiﬁcantly reduced CK-MB

Table 2

Hemodynamic parameters changes.

Groups Δ1 Change Δ2 Change Δ3 Change

HR SAP HR SAP HR SAP

Control 11.0475.3 11.7473.1 33.6477.8 17.174 56.05710.7 33.2775.8

AVP0.03 7.5178 1373.9 18.74711.3 23.377.2 8.55717.3 25.774.8

Atr 22.976.2 8.876.7 25711.9 27.177.6 19.877.7 32.476.3

AVPþAtr 4.5878.1 15.178 16.9717.7 27.978.9 10.14711.3 48.6712.5

CsA 0.5779.3 22.377.2 12.42712.4 21.277.9 29.9715.7 37.776.3

AVPþCsA 0.52713.2 16.172.5 1.6577.7 16.874.9 10.49712.3 27.973.6

The values are mean7S.E.M. HR, heart rate (beats/min); SAP, systolic arterial pressure (mmHg); AVP, arginine vasopressin; Atr, Atractyloside; CsA, Cyclosporine A.Δ1 Changes: differences between end of ischemia 30 and end of baseline period,Δ2 changes: differences between end of reperfusion 60 and end of baseline period,Δ3 changes:

differences between end of reperfusion 110 and end of baseline period.

Table 3

Intraventricular hemodynamic parameters at 10 min of end reperfusion.

Groups LVDP LVEDP RPP þdp/dt dp/dt

Control 7475 4.070.8 17,53971784 20297147 16387127 AVP0.03 8177 1.670.4 23,61672971 21957254 19687236 Atr 9079 6.271.2 33,06873722^a 25577189 25487160^b AVPþAtr 80711 3.870.3 26,86474190 22867325 21557317

CsA 9673 3.870.9 28,91872357 26047136 24187155

AVPþCsA 9376 4.170.8 30,54572771^b 25687159 23817165 The values are mean7S.E.M; AVP, arginine vasopressin; Atr, Atractyloside; CsA, Cyclosporine A; LVDP, left ventricular developed pressure (mmHg); LVEDP, left ventricle end-diastolic pressure (mmHg); RPP, rate pressure product (beats/

min mmHg 10³); the maximum rise and fall of LV pressures (þdp/dtanddp/

dt, respectively) (mmHg/s).

aPo0.01 vs. Control.

bPo0.05 vs. Control.

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level as compared to control group. Atractyloside and Cyclosporine A infusion in AVPþAtr and AVPþCsA groups signiﬁcantly returned LDH and CK-MB plasma level as seen as in control group (Table 4).

3.3.2. Lipid peroxidation level

MDA plasma level in AVP 0.03 group signiﬁcantly declined compared to control group. Administration of Atractyloside and Cyclosporine A prior to reperfusion in AVPþAtr and AVPþCsA groups returned MDA plasma level as shown in control (Table 4).

3.4. PAB assay

The PAB value of control group was 46.873 (HK unit) (Fig. 3).

Compared to control group, administration of AVP 0.03mg/rat (11.170.5 HK,Po0.001) could prevent the elevation of PAB value in plasma after ischemia/reperfusion injury and Compared to AVP 0.03 group, Atractyloside and Cyclosporine A in AVPþAtr (45.974.6 HK, Po0.001) and AVPþCsA (47.471.9 HK,Po0.001) groups signiﬁcantly increased PAB value and returned its level as seen as in control group.

3.5. Mitochondrial swelling assays

To determine whether AVP can modulate MPTP opening, we tested the effect of AVP on the decrease of A520 in isolated

mitochondria. This was used to indicate swelling of the mitochon- drion as a result of opening of the pore. At 200mmol/L, CaCl2, which is known to open the pore, evoked a large decrease inA520. This effect was inhibited by 1mmol/L CsA, conﬁrming that the decrease in absorbance was due to pore opening. Interestingly, AVP0.03mg/

rat in AVP0.03 group also inhibited the decrease inA520, suggesting that it may protect the heart by inhibiting pore opening (Fig. 4).

4. Discussion

In the previous work, we showed that AVP can induce preconditioning in a dose-dependent manner via V1 receptor (Nazari et al., 2011).

The present study revealed that exogenous AVP (0.03

μ

^{g/rat) can}

induce preconditioning and signiﬁcantly decreased infarct size, Biochemical parameters (LDH, CK-MB and MDA plasma levels) and PAB as compared to control group and Administration of Atractylo- side (as MPTP opener), decreased the cardioprotective effects of AVP.

It seems that the cardioprotective effect of AVP against myocardial infarction was mediated via inhibiting of MPTP permeability. Many studies have shown a role for mitochondria in reperfusion injury (Rajesh et al., 2003). The mitochondrial permeability transition pore (MPTP) is transmembrane protein that play a major role in both necrotic and apoptotic cell death. The major components of the MPTP are the adenine nucleotide translocase (ANT) in the inner membrane of the mitochondria, cyclophilin D in

Fig. 2.Myocardial area at risk (AAR/LV %) and infarct size (IS/AAR %) in Control, AVP (0.03mg/rat), Atr, AVP (0.03mg/rat)þAtr, CsA and AVP (0.03mg/rat)þCsA groups.

Data are presented as mean7S.E.M. AVP, arginine vasopressin; Atr, Atractyloside;

CsA, Cyclosporine A. ⁿⁿPo0.01, ⁿⁿⁿPo0.001 vs. control group. ^# Po0.05 vs.

AVP0.03 (n¼13).

Table 4

The plasma levels of LDH, CK-MB and MDA at the end of reperfusion in Control, AVP (0.03mg/rat), Atr, AVP (0.03mg/rat)þAtr, CsA and AVP (0.03mg/rat)þCsA groups.

Groups LDH (IU/dl) CK-MB (IU/dl) MDA (nmol/ml)

Control 367723.3 27.972.9 4.170.2

AVP0.03 182721.1^a 9.7871.2^b 2.170.3^a

Atr 6727124.8 14.971.2^c 2.770.4^a

AVPþAtr 4537103.5^d 28.677.4^e 3.070.4

CsA 460766.6 18.871.9 3.670.6

AVPþCsA 6357106.9^f 38.778.2^f 3.471.4

The values are expressed as mean7S.E.M; AVP, arginine vasopressin; Atr, Atracty- loside; CsA, Cyclosporine A; LDH, lactate dehydrogenase; CK-MB, creatine kinase- MB; MDA, malondialdehyde.

aPo0.01 vs. Control.

bPo0.001 vs. Control.

cPo0.05 vs. Control.

dPo0.05 vs. AVP0.03.

ePo0.01 vs. AVP0.03.

fPo0.001 vs. AVP0.03.

Fig. 3.The PAB value of Control, AVP (0.03mg/rat), Atr, AVP (0.03mg/rat)þAtr, CsA and AVP (0.03mg/rat)þCsA groups. AVP, arginine vasopressin; Atr, Atractyloside;

CsA, Cyclosporine A. Data are presented as mean7S.E.M.ⁿⁿⁿPo0.001 vs. control group.^###Po0.001 vs. AVP0.03 group (n¼13).

Fig. 4.Percentage reduction in the rate of absorption at 520 nm (A520) in Control–

CaCl2, CsA–CaCl2and AVP–CaCl2groups in suspensions of rat heart mitochondria exposed to 200 mmol/L CaCl2. Mitochondria from control group that treated with 200 mmol/L CaCl2 served as the Control–CaCl2. Absorbance data 21 min after addition of 200 mmol/L CaCl2. Data are the mean7S.E.M.ⁿPo0.05 compared with Control–CaCl2(n¼4).

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the matrix and the voltage dependent anion channel (VDAC) in the outer membrane. These proteins are thought to come together at intermembrane junctions to form the MPTP (Halestrap and Brenner, 2003). Under normal physiological conditions, the mitochondrial inner membrane is impermeable. However, under stress, a nonspeciﬁc pore known as the MPTP can open in the mitochondrial inner membrane that allows free passage of any molecule of o1.5 kDa (Halestrap et al., 2004). The key factor responsible for MPTP opening is mitochondrial calcium overload, especially when this is accompanied by oxidative stress, adenine nucleotide deple- tion, elevated phosphate concentrations, and mitochondrial depo- larization (Halestrap et al., 2004).

Increased free oxygen radicals cause tissue damage through the peroxidation of the lipids present in the cell membranes and increasing lipid peroxidation might be used as a sign of the tissue damage. MDA is thefinal product of lipid peroxidation and is used as an indicator of tissue damage caused by oxygen free radicals (Kim et al., 2000; Molina and Garcia, 1997; Sahin et al., 2011). Current data show that plasma level of MDA significantly reduced following exogenous administration of AVP compared to control group moreover caused decrease of the PAB value in favor of the antioxidant status and Atractyloside in AVPþAtr group significantly increased PAB value and returned its level to oxidant status as seen as in control group. A significant amount of evidence is present in the literature to support the role of oxygen free radicals in pathogenesis of I/R injury (Das et al., 1986; Tosaki et al., 1993). This receives further support from the evidence that a variety of free radical scavengers and antioxidants are capable of ameliorating I/R injury (Das and Maulik, 1994). In addition, it has been demonstrated that NO plays a crucial role in cardiac preconditioning. NO production was associated with myocardial preservation during ischemia (Imani et al., 2011; Maulik et al., 1995).Martinez et al. (2003)reported that the modulatory role of NO in the coronary response to AVP may be preserved during partial coronary occlusion in anesthetized goats. Therefore, it seems that AVP via NO production could induce cardioprotection against I/R injury.

Many studies have shown a role for mitochondria in reperfusion injury following ischemia. During reperfusion, cellular calcium increases, resulting in opening of the MPTP and release of cytochrome C. Subsequent activation of caspases results in the failure of ATP generation and mitochondrial membrane potential and leads to cell death (Rajesh et al., 2003). Studies have shown that IPC inhibits a process occurring during ischemia and reperfusion that is responsible for sensitizing the MPTP to calcium and reduced MPTP opening at the onset of reperfusion and exerts its protective effects (Halestrap et al., 2007). So it seems that MPTP is one of the major pathways and main effectors in cardioprotection following precondition period. Atractyloside induces pore formation by ANT, and results in permeabilization of MPTP, causing uncoupling of oxidative phosphorylation (Xu et al., 2001) and abolished the beneﬁcial effect of preconditioning (Hausenloy and Yellon, 2007). The current study has shown that AVP owns a cardioprotective effect via myocyte mitochondria against I/R injury in rat in vivo.

The present study has shown that Cyclosporine A similar to AVP signiﬁcantly reduced infarct size vs. control group but in other factors included: biochemical parameters (LDH, CK-MB and MDA) and PAB value, we did not see any cardioprotective effects. Moreover CsA caused increase of the PAB value in favor of the oxidant status.

Cyclosporine A (CsA) is a well-known immunosuppressant, but also a potent inhibitor of MPTP opening and is considered to be inherently protective in conditions of ischemia, e.g. in cardiac tissue.

There are several hypotheses to explain the mechanism of CsA- induced adverse effects, including the formation of free oxygen radicals, lipid peroxidation and induction of the cytochrome P450 system (Bianchi et al., 2003; Wang and Salahudeen, 1995; Zachariae, 1999). Several reports have suggested that oxidative stress is the most

possible pathway of CsA toxicity (Grieve and Shah, 2003; Rezzani et al., 2003).

CsA due to MPTP inhibition regardless of its oxidant properties has been used in heart ischemia–reperfusion studies and had more beneﬁcial effects (as MPTP inhibitor). In other study CsA (10 mg/kg) was given 10 min prior to ischemia via the femoral vein. CsA signiﬁcantly reduced infarct size and decreased caspase- 3 activity in the myocardium and relieved the injury of mitochondria. CsA reduced the cardiac damage associated with ischemia– reperfusion injury of the heart. The cardioprotective effects of CsA might be associated with the protection of mitochondria and the inhibition of caspase-3 activity (Xie and Yu, 2007).

CsA caused SAP not only decreased at the end 60 min reperfusion but also signiﬁcantly increased that compared to control group. Rezzani et al. had shown that CsA had adverse effect including increased blood pressure (Rezzani, 2004) that was mediated via increased synthesis of vasoconstrictor eicosanoids (Bianchi et al., 2003). Exogenous AVP in both groups of (AVPþAtr) and (AVPþCsA) prevented the decrease in HR at the end of reperfusion compared to their baseline. It seems that this effect not related to opening and closing of MPTP and probably is done by other pathways that remain to be explored in future studies.

Opening of the MPTP may result in mitochondrial swelling, collapse of mitochondrial membrane potential, uncoupling of mitochondrial oxidative phosphorylation and cytochrome C release, leading to both necrosis and apoptosis. It was demonstrated that modulation of MPTP opening has been observed in cardioprotection by both preconditioning and post-conditioning of the heart (Liu et al., 2008). Mitochondria isolated from AVP–CaCl2group showed reduced swelling to challenge with 200mmol/L CaCl2. Furthermore, these mitochondria showed a degree of swelling similar to that induced by CsA. Therefore, it may be suggested that AVP owns a cardioprotective effect against I/R injury by inhibiting MPTP opening. Although the mechanism of the action of AVP in preconditioning responses was not completely explored in my previous (Nazari et al., 2011) and present study, but it had shown that mainly direct activation of V1 receptors on cardiac myocyte can active intracellular signaling specially inhibiting MPTP opening and events previously described (Briley et al., 1994; Dayanithi et al., 2008) similar to preconditioning signaling (Starkopf et al., 1998). Another possibility to consider is that AVP reduced free radical formation in the hearts and ﬁnally by inhibiting MPTP opening on cardiac tissue may be the cause of its protection. Thus, AVP may induce preconditioning via several different mechanisms. However, the exact mechanism(s) behind the cardioprotective effects of AVP remain to be explored in future studies.

Further studies are needed to truly disentangle the cardioprotective mechanism of AVP against ischemia–reperfusion injury that included:

a- The cardioprotective effect of endogenous vasopressin (AVP) against ischemia–reperfusion injuries in the anesthetized rat heart.

b- Myocardial expression of V1 receptor in condition of ischemia– reperfusion injuries.

c- Examined the relationship between oxytocin receptors and vasopressin in condition of heart ischemia–reperfusion.

d- The molecular mechanisms of mitochondria responsible for the cardioprotective effect of AVP such as: apoptotic factors expression, myocardial ATP content, the release of cytochrome c, etc.

5. Conclusion

The present study exhibited that exogenous AVP had hormonal preconditioning effect mainly via antioxidant effect and probably by inhibiting MPTP opening on cardiac myocyte against ischemia/

reperfusion injury in rat heart in vivo.

(7)

Conﬂict of interest None declared.

Acknowledgments

This study was supported ﬁnancially by Tehran University of Medical Science.

References

Alamdari, D.H., Paletas, K., Pegiou, T., Sarigianni, M., Befani, C., Koliakos, G., 2007. A novel assay for the evaluation of the prooxidant–antioxidant balance, before and after antioxidant vitamin administration in type II diabetes patients. Clin.

Biochem. 40, 248–254.

Argaud, L., Gateau-Roesch, O., Raisky, O., Loufouat, J., Robert, D., Ovize, M., 2005.

Postconditioning inhibits mitochondrial permeability transition. Circulation 111, 194–197.

Baines, C.P., Song, C.X., Zheng, Y.T., Wang, G.W., Zhang, J., Wang, O.L., Guo, Y., Bolli, R., Cardwell, E.M., Ping, P., 2003. Protein kinase Cepsilon interacts with and inhibits the permeability transition pore in cardiac mitochondria. Circ. Res. 92, 873–880.

Bianchi, R., Rodella, L., Rezzani, R., 2003. Cyclosporine A up-regulates expression of matrix metalloproteinase 2 and vascular endothelial growth factor in rat heart.

Int. Immunopharmacol. 3, 427–433.

Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding.

Anal. Biochem. 72, 248–254.

Briley, E.M., Lolait, S.J., Axelrod, J., Felder, C.C., 1994. The cloned vasopressin V1a receptor stimulates phospholipase A2, phospholipase C, and phospholipase D through activation of receptor-operated calcium channels. Neuropeptides 27, 63–74.

Das, D.K., Engelman, R.M., Rousou, J.A., Breyer, R.H., Otani, H., Lemeshow, S., 1986.

Pathophysiology of superoxide radical as potential mediator of reperfusion injury in pig heart. Basic Res. Cardiol. 81, 155–166.

Das, D.K., Maulik, N., 1994. Antioxidant effectiveness in ischemia–reperfusion tissue injury. Methods Enzymol. 233, 601–610.

Dayanithi, G., Viero, C., Shibuya, I., 2008. The role of calcium in the action and release of vasopressin and oxytocin from CNS neurones/terminals to the heart.

J. Physiol. Pharmacol.: Off. J. Pol. Physiol. Soc. 59 (Suppl. 8), S7–S26.

Dunser, M.W., Mayr, A.J., Stallinger, A., Ulmer, H., Ritsch, N., Knotzer, H., Pajk, W., Mutz, N.J., Hasibeder, W.R., 2002. Cardiac performance during vasopressin infusion in postcardiotomy shock. Intensive Care Med. 28, 746–751.

Ghazi-Khansari, M., Mohammadi-Bardbori, A., 2007. Captopril ameliorates toxicity induced by paraquat in mitochondria isolated from the rat liver. Toxicol.

In Vitro: Int. J. Publ. Assoc. BIBRA 21, 403–407.

Grieve, D.J., Shah, A.M., 2003. Oxidative stress in heart failure. More than just damage. Eur. Heart J. 24, 2161–2163.

Grifﬁths, E.J., Halestrap, A.P., 1993. Protection by Cyclosporin A of ischemia/

reperfusion-induced damage in isolated rat hearts. J. Mol. Cell. Cardiol. 25, 1461–1469.

Halestrap, A.P., Brenner, C., 2003. The adenine nucleotide translocase: a central component of the mitochondrial permeability transition pore and key player in cell death. Curr. Med. Chem. 10, 1507–1525.

Halestrap, A.P., Clarke, S.J., Javadov, S.A., 2004. Mitochondrial permeability transition pore opening during myocardial reperfusion–a target for cardioprotection. Cardiovasc. Res. 61, 372–385.

Halestrap, A.P., Clarke, S.J., Khaliulin, I., 2007. The role of mitochondria in protection of the heart by preconditioning. Biochim. Biophys. Acta 1767, 1007–1031.

Hausenloy, D.J., Yellon, D.M., 2007. Preconditioning and postconditioning: united at reperfusion. Pharmacol. Ther. 116, 173–191.

Holmes, C.L., Patel, B.M., Russell, J.A., Walley, K.R., 2001. Physiology of vasopressin relevant to management of septic shock. Chest 120, 989–1002.

Imani, A., Faghihi, M., Sadr, S.S., Niaraki, S.S., Alizadeh, A.M., 2011. Noradrenaline protects in vivo rat heart against infarction and ventricular arrhythmias via nitric oxide and reactive oxygen species. J. Surg. Res. 169, 9–15.

Kim, H.S., Kwack, S.J., Lee, B.M., 2000. Lipid peroxidation, antioxidant enzymes, and benzo[a]pyrene-quinones in the blood of rats treated with benzo[a]pyrene.

Chem. Biol. Interact. 127, 139–150.

Lim, W., Whitlock, R., Khera, V., Devereaux, P.J., Tkaczyk, A., Heels-Ansdell, D., Jacka, M., Cook, D., 2010. Etiology of troponin elevation in critically ill patients. J. Crit.

Care 25, 322–328.

Liu, Y.N., Zhou, Z.M., Chen, P., 2008. Evidence that hydroxysafﬂor yellow A protects the heart against ischaemia–reperfusion injury by inhibiting mitochondrial permeability transition pore opening. Clin. Exp. Pharmacol. Physiol. 35, 211–216.

Martinez, M.A., Fernandez, N., Climent, B., Garcia-Villalon, A.L., Monge, L., Sanz, E., Dieguez, G., 2003. Coronary effects of vasopressin during partial ischemia and reperfusion in anesthetized goats. Role of nitric oxide and prostanoids. Eur. J.

Pharmacol. 473, 55–63.

Maulik, N., Engelman, D.T., Watanabe, M., Engelman, R.M., Maulik, G., Cordis, G.A., Das, D.K., 1995. Nitric oxide signaling in ischemic heart. Cardiovasc. Res. 30, 593–601.

Molina, H., Garcia, M., 1997. Enzymatic defenses of the rat heart against lipid peroxidation. Mech. Ageing Dev. 97, 1–7.

Nazareth, W., Yafei, N., Crompton, M., 1991. Inhibition of anoxia-induced injury in heart myocytes by cyclosporin A. J .Mol. Cell. Cardiol. 23, 1351–1354.

Nazari, A., Sadr, S.S., Faghihi, M., Imani, A., Moghimian, M., 2011. The cardioprotective effect of different doses of vasopressin (AVP) against ischemia–reperfusion injuries in the anesthetized rat heart. Peptides 32, 2459–2466.

Okamura, T., Ayajiki, K., Fujioka, H., Toda, N., 1999. Mechanisms underlying arginine vasopressin-induced relaxation in monkey isolated coronary arteries. J. Hyper- tens. 17, 673–678.

Pelletier, J.S., LaBossiere, J., Dicken, B., Gill, R.S., Sergi, C., Tahbaz, N., Bigam, D., Cheung, P.Y., 2013. Low-dose vasopressin improves cardiac function in newborn piglets with acute hypoxia-reoxygenation. Shock 40, 320–326.

Rajesh, K.G., Sasaguri, S., Zhitian, Z., Suzuki, R., Asakai, R., Maeda, H., 2003. Second window of ischemic preconditioning regulates mitochondrial permeability transition pore by enhancing Bcl-2 expression. Cardiovasc. Res. 59, 297–307.

Rezzani, R., 2004. Cyclosporine A and adverse effects on organs: histochemical studies. Prog. Histochem. Cytochem. 39, 85–128.

Rezzani, R., Rodella, L., Dessy, C., Daneau, G., Bianchi, R., Feron, O., 2003. Changes in Hsp90 expression determine the effects of cyclosporine A on the NO pathway in rat myocardium. FEBS Lett. 552, 125–129.

Sahin, M.A., Yucel, O., Guler, A., Doganci, S., Jahollari, A., Cingoz, F., Arslan, S., Gamsizkan, M., Yaman, H., Demirkilic, U., 2011. Is there any cardioprotective role of Taurine during cold ischemic period following global myocardial ischemia? J. Cardiothorac. Surg. 6, 31.

Sastre, J., Pallardo, F.V., Vina, J., 2003. The role of mitochondrial oxidative stress in aging. Free Radic. Biol. Med. 35, 1–8.

Schuh, J., Fairclough Jr., G.F., Haschemeyer, R.H., 1978. Oxygen-mediated hetero- geneity of apo-low-density lipoprotein. Proc. Natl. Acad. Sci. USA 75, 3173–3177.

Smith, S.M., Myers, J.H., Kaplan, H.M., 1979. Catheterization of the left cardiac ventricle of the rat. Lab. Anim. 13, 15–16.

Starkopf, J., Andreasen, T.V., Bugge, E., Ytrehus, K., 1998. Lipid peroxidation, arachidonic acid and products of the lipoxygenase pathway in ischaemic preconditioning of rat heart. Cardiovasc. Res. 37, 66–75.

Tosaki, A., Bagchi, D., Pali, T., Cordis, G.A., Das, D.K., 1993. Comparisons of ESR and HPLC methods for the detection of OH. radicals in ischemic/reperfused hearts. A relationship between the genesis of free radicals and reperfusion arrhythmias.

Biochem. Pharmacol. 45, 961–969.

Wang, C., Salahudeen, A.K., 1995. Lipid peroxidation accompanies cyclosporine nephrotoxicity: effects of vitamin E. Kidney Int. 47, 927–934.

Wang, G., Liem, D.A., Vondriska, T.M., Honda, H.M., Korge, P., Pantaleon, D.M., Qiao, X., Wang, Y., Weiss, J.N., Ping, P., 2005. Nitric oxide donors protect murine myocardium against infarction via modulation of mitochondrial permeability transition. Am. J. Physiol. Heart Circ. Physiol. 288, H1290–H1295.

Xie, J.R., Yu, L.N., 2007. Cardioprotective effects of cyclosporine A in an in vivo model of myocardial ischemia and reperfusion. Acta Anaesthesiol. Scand. 51, 909–913.

Xu, M., Wang, Y., Hirai, K., Ayub, A., Ashraf, M., 2001. Calcium preconditioning inhibits mitochondrial permeability transition and apoptosis. Am. J. Physiol.

Heart Circ. Physiol. 280, H899–H908.

Yang, X.M., Proctor, J.B., Cui, L., Krieg, T., Downey, J.M., Cohen, M.V., 2004. Multiple, brief coronary occlusions during early reperfusion protect rabbit hearts by targeting cell signaling pathways. J. Am. Coll. Cardiol. 44, 1103–1110.

Zachariae, H., 1999. Renal toxicity of long-term cyclosporin. Scand. J. Rheumatol. 28, 65–68.

Zhao, Y., Ye, L., Liu, H., Xia, Q., Zhang, Y., Yang, X., Wang, K., 2010. Vanadium compounds induced mitochondria permeability transition pore (PTP) opening related to oxidative stress. J. Inorg. Biochem. 104, 371–378.

Zhu, W., Tilley, D.G., Myers, V.D., Coleman, R.C., Feldman, A.M., 2013. Arginine vasopressin enhances cell survival via a G protein-coupled receptor kinase 2/

beta-arrestin1/extracellular-regulated kinase 1/2-dependent pathway in H9c2 cells. Mol. Pharmacol. 84, 227–235.

Zoratti, M., Szabo, I., 1995. The mitochondrial permeability transition. Biochim.

Biophys. Acta 1241, 139–176.